Combination vaccines with low dose of hib conjugate

ABSTRACT

Provided herein are combination vaccines comprising antigens for protecting a subject against at least diphtheria, tetanus, pertussis and Hib, wherein: (a) the antigen for protecting against Hib is a conjugate of a Hib capsular saccharide; (b) the concentration of the Hib conjugate in the vaccine is &lt;15 μg/ml; and (c) the Hib conjugate has never been lyophilised.

All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

This invention is in the field of combination vaccines, particularlythose for protecting against diphtheria, tetanus, pertussis and H.influenzae type b (‘Hib’).

BACKGROUND ART

Combination vaccines including antigens for immunizing againstdiphtheria, tetanus, pertussis and Hib are known (‘DTP-Hib’ vaccines).Three such vaccines have been marketed under the names TETRAMUNE™ andQUATTVAXEM™ (which use cellular pertussis antigens ‘DTwP-Hib’) andINFANRIX-Hib™ (which uses acellular pertussis antigens ‘DTaP-Hib’).

The inclusion of Hib-conjugate components in DTaP-Hib vaccines has beenassociated with reductions in the anti-Hib response [1, 2]. Furthermore,Hib-conjugates are unstable in aqueous media and cannot surviveprolonged storage in this form [3]. For this reason, in combinationvaccines that include Hib-conjugate antigens, it is common for the Hibcomponent to be provided as a lyophilised powder that is reconstitutedat the time of delivery with a liquid formulation of the other antigens.

Hib-conjugate antigens are not cheap to produce, and there is concernthat their cost will inhibit widespread use in developing countries, andso alternative strategies for Hib-conjugate use have been developed[4-6]. One approach to furthering their use has been to give two doses(e.g. at 3 & 5 months of age [5] or at 4 & 6 months [6]), rather thanthe normal three doses (2, 4 & 6 months [7]). In a different approach,whereas Hib-conjugates are typically given at 10 μg/dose, lower doses(typically fractions e.g. ½, ⅓, ¼, etc.) have been used [4,6]. In ref.6, for example, Hib-conjugates were administered at 5 μg/dose or 3.33μg/dose.

The same approach has been extended to Hib-conjugates within DTP-Hibvaccines. For example, reference 8 compares full-dose, half-dose andthird-dose use of Hib-conjugate in combination with a DTwP vaccine and,although geometric mean concentrations of anti-PRP bodies were reducedin patients receiving combined DTP-Hib vaccines compared to separateadministration of DTP and Hib, acceptable protective anti-Hib immuneresponses were seen in all cases. Reference 9 uses a 10-fold dilution ofHib-conjugate dosage by reconstituting a single Hib dose with a ten-dosevial of DTwP. Reference 10 discloses reconstitution of lyophilisedHib-conjugate at full dose, half-dose or quarter-dose using theTRITANRIX™ DTwP-HBsAg vaccine.

In each of these cases, however, the Hib-conjugate was in lyophilisedform and had to be reconstituted by aqueous DTP antigens prior toadministration. These vaccines must thus be provided in two separatecontainers (aqueous DTP in one, lyophilised Hib in the other), and thisdouble container requirement imposes additional cost and logisticalrequirements at the packaging stage, the transport stage, the storagestage and the administration stage. As the reduced-dose vaccines areintended to reduce cost and encourage distribution in the developingworld then these additional requirements are significant disadvantages.The requirement for a reconstitution step also means that there is arisk of error by the end user, a tendency towards non-standardiseddosages, a risk of contamination of the mixed product and a need totrain staff in the reconstitution procedure. All of these problemsfrustrate the intended target market i.e. the developing world.

There thus remains a need for a combination vaccine that includes a lowdose of Hib-conjugate antigen and does not need separate packaging ofthe Hib antigen.

DISCLOSURE OF THE INVENTION

The invention provides a combination vaccine comprising antigens forprotecting a subject against at least diphtheria (‘D’), tetanus (‘T’),pertussis (‘P’) and H. influenzae type b (‘Hib’), wherein: (a) theantigen for protecting against Hib is a conjugate of a Hib capsularsaccharide; (b) the concentration of the Hib conjugate in the vaccine is<15 μg/ml; and (c) the Hib conjugate has never been lyophilised.Vaccines of the invention have been found to be safe and to be highlyimmunogenic when compared to the immune responses observed in references6, 8 and 9.

The invention also provides a combination vaccine comprising antigensfor protecting a subject against at least diphtheria (‘D’), tetanus(‘T’), pertussis (‘P’) and H. influenzae type b (‘Hib’), wherein: (a)the antigen for protecting against Hib is a conjugate of a Hib capsularsaccharide; (b) the concentration of the Hib conjugate in the vaccine is<15 μg/ml; and (c) the vaccine (i) does not contain an aluminiumhydroxide adjuvant and/or (ii) does not contain an aluminium potassiumsulfate adjuvant. Aluminium hydroxide is believed to be involved indegradation of Hib saccharide conjugates, and so as an adjuvant thevaccine preferably includes an aluminium phosphate adjuvant instead.Where an aluminium adjuvant (e.g. an aluminium phosphate adjuvant) ispresent, it is preferred that the Hib conjugate is not adsorbed to it.

The invention also provides a vial having a piercable seal andcontaining a combination vaccine, which combination vaccine comprisesantigens for protecting a subject against at least diphtheria, tetanus,pertussis and H. influenzae type b (‘Hib’), wherein the antigen forprotecting against Hib is a conjugate of a Hib capsular saccharide, andwherein: (a) the concentration of the Hib conjugate in the vaccine is<15 μg/ml, and (b) the vial's piercable seal has not been pierced.

The invention also provides a hermetically-sealed container containing acombination vaccine comprising antigens for protecting a subject againstat least diphtheria, tetanus, pertussis and H. influenzae type b(‘Hib’), wherein the antigen for protecting against Hib is a conjugateof a Hib capsular saccharide, and wherein the concentration of the Hibconjugate in the vaccine is <15 μg/ml.

The invention also provides a process for preparing a combinationvaccine comprising antigens for protecting a subject against at leastdiphtheria (‘D’), tetanus (‘T’), pertussis (‘P’) and H. influenzae typeb (‘Hib’), wherein the antigen for protecting against Hib is a conjugateof a Hib capsular saccharide and the concentration of Hib conjugate inthe vaccine is <15 μg/ml, and wherein (a) the process comprises a stepof admixing said antigens for protecting against D, T, P and Hib, and(b) the process (i) does not include a step of lyophilisation of the Hibconjugate antigen, and/or (ii) does not include a step of packaging thediphtheria, tetanus and pertussis antigens in admixed form separatelyfrom the Hib conjugate antigen.

The invention also provides a process for inserting a combinationvaccine into a container, wherein: (a) the vaccine comprises antigensfor protecting a subject against at least diphtheria, tetanus, pertussisand H. influenzae type b (‘Hib’); (b) the antigen for protecting againstHib is a conjugate of a Hib capsular saccharide; and (c) theconcentration of the Hib conjugate in the vaccine is <15 μg/ml.

The invention also provides a process for attaching a label to acontainer, wherein: (a) the container contains a combination vaccinethat comprises antigens for protecting a subject against at leastdiphtheria, tetanus, pertussis and H. influenzae type b (‘Hib’); (b) theantigen for protecting against Hib is a conjugate of a Hib capsularsaccharide; and (c) the concentration of the Hib conjugate in thevaccine is <15 μg/ml. The label may indicate that the container containsa vaccine.

The invention also provides a process for inserting a combinationvaccine into a container and then extracting the vaccine from thecontainer, wherein: (a) the vaccine comprises antigens for protecting asubject against at least diphtheria, tetanus, pertussis and H.influenzae type b (‘Hib’); (b) the antigen for protecting against Hib isa conjugate of a Hib capsular saccharide; and (c) the concentration ofthe Hib conjugate in the vaccine is <15 μg/ml.

DTP Components

The diphtheria antigen is preferably a diphtheria toxoid. Thepreparation of diphtheria toxoids is well documented [e.g. chapter 13 ofreference 11]. Any suitable diphtheria toxoid may be used. Theconcentration of diphtheria toxoid is generally between 5 and 100 Lf/ml.A preferred concentration is between 10 and 50 Lf/ml. A more preferredconcentration is between 20 and 40 Lf/ml. Most preferably, theconcentration is about 30 Lf/ml. As an alternative, a preferredconcentration is between 5 and 25 Lf/ml, a more preferred concentrationis between 10 and 20 Lf/ml, and a most preferred concentration is about15 Lf/ml. Where an acellular pertussis antigen is used, however, then apreferred concentration for the diphtheria toxoid is about 50 Lf/ml.

The tetanus antigen is preferably a tetanus toxoid. The preparation oftetanus toxoids is well documented [e.g. chapter 27 of reference 11].Any suitable tetanus toxoid may be used. The concentration of tetanustoxoid is generally between 1 and 50 Lf/ml. A preferred concentration isbetween 2 and 9 Lf/ml. A more preferred concentration is between 5 and 8Lf/ml. Most preferably, the concentration is about 6.5 Lf/ml. Where anacellular pertussis antigen is used, however, then a preferredconcentration for the diphtheria toxoid is about 20 Lf/ml.

The pertussis antigen used according to the invention may be cellular(e.g. whole cell) or acellular. The preparation of both types of antigenis well documented [e.g. see chapter 21 of reference 11; see alsoreference 12]. For cellular pertussis antigens, the concentration ofpertussis antigens is generally between 5 and 50 OU/ml. A preferredconcentration is between 10 and 40 OU/ml. A more preferred concentrationis between 25 and 350 U/ml. Most preferably, the concentration is about30 OU/ml. Where acellular antigens are used, it is preferred to usepertussis holotoxin (PT) and filamentous haemagglutinin (FHA), morepreferably combined with pertactin (also known as PRN or 69 kDa antigen)and, optionally, agglutinogens (also known as fimbriae) 2 and 3 [13].Typical levels of pertussis antigens per vaccine dose (e.g. per 0.5 ml)are: 10 μg PT, 5 μg FHA, 3 μg or 5 μg PRN, 5 μg combined fimbriae. PT isa toxic protein and, when present in the pertussis antigen, it ispreferably detoxified. Detoxification may be by chemical and/or geneticmeans. A preferred detoxified mutant is the 9K/129G double mutant [14].

The Hib Conjugate

The H. influenzae type B antigen used in vaccines of the inventioncomprises a Hib capsular saccharide antigen. Saccharide antigens from H.influenzae b are well known [e.g. chapter 14 of ref. 11]. The Hibsaccharide is conjugated to a carrier protein in order to enhance itsimmunogenicity, especially in children. The preparation of Hib capsularsaccharide is well documented [e.g. references 15 to 24]. The inventionmay use any suitable Hib conjugate. Suitable carrier proteins aredescribed above, and preferred carriers for Hib saccharides are CRM₁₉₇(‘HbOC’), tetanus toxoid (‘PRP-T’) and the outer membrane complex of N.meningitidis (‘PRP-OMP’).

The saccharide moiety of the conjugate may be a polysaccharide (e.g.full-length polyribosylribitol phosphate (PRP)), but it is preferred touse oligosaccharides (e.g. MW from ˜1 to ˜5 kDa). These are convenientlyformed by fragmentation of purified PRP (e.g. by hydrolysis), which willusually be followed by purification of the fragments of the desiredsize. Where the composition of the invention includes a conjugatedoligosaccharide, oligosaccharide preparation should precede conjugation.

Preferred carrier proteins for covalent conjugation are bacterial toxinsor toxoids, such as diphtheria toxoid or tetanus toxoid. The CRM₁₉₇diphtheria toxin mutant [25-27] is particularly preferred. Othersuitable carrier proteins include the N. meningitidis outer membraneprotein [28], synthetic peptides [29, 30], heat shock proteins [31, 32],pertussis proteins [33, 34], cytokines [35], lymphokines [35], hormones[35], growth factors [35], artificial proteins comprising multiple humanCD4⁺ T cell epitopes from various pathogen-derived antigens [36],protein D from H. influenzae [37, 38], pneumococcal surface protein PspA[39], iron-uptake proteins [40], toxin A or B from C. difficile [41],etc. Preferred carriers are diphtheria toxoid, tetanus toxoid andCRM₁₉₇.

Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e.excess protein) and 5:1 (i.e. excess saccharide) may be used e.g. ratiosbetween 1:2 and 5:1 and ratios between 1:1.25 and 1:2.5.

Conjugates may be used in conjunction with free carrier protein [42].When a given carrier protein is present in both free and conjugated formin a composition of the invention, the unconjugated form is preferablyno more than 5% of the total amount of the carrier protein in thecomposition as a whole, and more preferably present at less than 2% byweight.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

The saccharide will typically be activated or functionalised prior toconjugation. Activation may involve, for example, cyanylating reagentssuch as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate[43, 44, etc.]). Other suitable techniques use carbodiimides,hydrazides, active esters, norborane, p-nitrobenzoic acid,N-hydroxysuccinimide, S-NHS, EDC, TSTU; see also the introduction toreference 22).

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 45 and 46. One type oflinkage involves reductive amination of the polysaccharide, coupling theresulting amino group with one end of an adipic acid linker group, andthen coupling a protein to the other end of the adipic acid linker group[20, 47, 48]. Other linkers include B-propionamido [49],nitrophenyl-ethylamine [50], haloacyl halides [51], glycosidic linkages[52], 6-aminocaproic acid [53], ADH [54], C₄ to C₁₂ moieties [55] etc.As an alternative to using a linker, direct linkage can be used. Directlinkages to the protein may comprise oxidation of the polysaccharidefollowed by reductive amination with the protein, as described in, forexample, references 56 and 57.

A process involving the introduction of amino groups into the saccharide(e.g. by replacing terminal ═O groups with —NH₂) followed byderivatisation with an adipic diester (e.g. adipic acidN-hydroxysuccinimido diester) and reaction with carrier protein ispreferred. Another preferred reaction uses CDAP activation with aprotein D carrier.

After conjugation, free and conjugated saccharides can be separated.There are many suitable methods for this separation, includinghydrophobic chromatography, tangential ultrafiltration, diafiltration,etc. [see also refs. 58 & 59, etc.]. If a vaccine comprises a givensaccharide in both free and conjugated forms, the unconjugated form ispreferably no more than 20% by weight of the total amount of thatsaccharide in the composition as a whole (e.g. ≦15%, ≦10%, ≦5%, ≦2%,≦1%).

A preferred conjugate comprises a Hib oligosaccharide covalently linkedto CRM₁₉₇ via an adipic acid linker [60, 61]. Tetanus toxoid is also apreferred carrier.

Administration of the Hib antigen preferably results in an anti-PRPantibody concentration of ≧0.15 μg/ml, and more preferably ≧1 μg/ml.These are the standard acceptable response thresholds.

The concentration of Hib conjugate in vaccines of the invention is <15μg/ml e.g. ≦14 μg/ml, ≦12 μg/ml, ≦10 μg/ml, ≦7.5 μg/ml, ≦5 μg/ml, ≦4μg/ml, ≦3 μg/ml, ≦2 μg/ml, ≦1 μg/ml, etc. Where the carrier protein isnot OMPC then it is possible to use slightly higher doses e.g. <20μg/ml, ≦19 μg/ml, ≦18 μg/ml, ≦17 μg/ml, ≦16 μg/ml, etc. Theconcentration of Hib conjugate in vaccines of the invention willgenerally, however, be at least 0.1 μg/ml e.g. ≧0.2 μg/ml, ≧0.3 μg/ml,≧0.4 μg/ml, ≧0.5 μg/ml, ≧0.6 μg/ml, ≧0.7 μg/ml, ≧0.8 μg/ml, ≧0.9 μg/ml,≧1.0 μg/ml, ≧1.25 μg/ml, ≧1.5 μg/ml, ≧2.0 μg/ml, ≧3.5 μg/ml, etc. Thuspreferred ranges for the concentration of Hib conjugate in the vaccinesare from d₁ to d₂ μg/ml, where: (i) d₁<d₂; (ii) d₁ is selected from 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5 and 4.0; and (iii) d₂is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

Concentrations of Hib conjugates are defined in the description and theclaims in terms of mass of saccharide (i.e. the dose of the conjugate(carrier+saccharide) as a whole is higher than the stated dose) in orderto avoid variation due to choice of carrier.

Hib conjugate antigens in compositions of the invention are not and havenever been lyophilised.

Adjuvants

Aluminium hydroxide is believed to be involved in degradation of Hibsaccharide conjugates [62], and so as an adjuvant the vaccine preferablyincludes an aluminium phosphate adjuvant instead.

Where an aluminium phosphate adjuvant is present, it is preferred thatthe Hib conjugate is not adsorbed to it, in contrast to reference 63.Non-adsorption can be achieved during manufacture by selection of thecorrect mixing order, by selecting an appropriate pH duringantigen/adjuvant mixing, and/or by choosing an adjuvant with anappropriate point of zero charge (PZC) [64] (see below).

Where aluminium phosphate is present, diphtheria toxoid will typicallybe adsorbed onto aluminium phosphate. It is preferred that adsorption ispartial e.g. of the total diphtheria toxoid in the composition, about30-80% by weight is adsorbed (e.g. about 40%-70%, about 50%-60% etc.).Adsorption of diphtheria toxoid rises over time when stored at about 37°C. Tetanus toxoid will typically be adsorbed onto aluminium phosphate.It is preferred that adsorption is partial e.g. of the total tetanustoxoid in the composition, no more than 40% by weight is adsorbed (e.g.no more than 30%, no more than 20%, no more than 10% etc.). Levels oftetanus toxoid adsorption may be about 0%. Hib-conjugate remainsun-adsorbed to aluminium adjuvant. It is preferred that no more than 15%by weight of Hib-conjugate in the composition should be adsorbed toaluminium phosphate (e.g. at most 10%, at most 5%, at most 4%, at most3%, at most 2%, or at most 1%).

The term “aluminium phosphate” as used herein includes aluminiumphosphate, aluminium hydroxyphosphate and aluminium hydroxyphosphatesulfate. The preferred form of aluminium phosphate for use with thepresent invention is a hydroxyphosphate salt.

The PO₄/Al³⁺ molar ratio of the aluminium phosphate will generally bebetween 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably0.95±0.1. A typical adjuvant is amorphous aluminium hydroxyphosphatewith PO₄/Al molar ratio between 0.84 and 0.92, included at 0.6 mgAl³⁺/ml. The aluminium phosphate will generally be amorphous,particularly for hydroxyphosphate salts. The aluminium phosphate willgenerally be particulate. Typical diameters of the particles are in therange 0.5-20 μm (e.g. about 5-10 μm) after any antigen adsorption.

The PZC of aluminium phosphate is inversely related to the degree ofsubstitution of phosphate for hydroxyl, and this degree of substitutioncan vary depending on reaction conditions and concentration of reactantsused for preparing the salt by precipitation. PZC is also altered bychanging the concentration of free phosphate ions in solution (morephosphate=more acidic PZC) or by adding a buffer such as a histidinebuffer (makes PZC more basic). Aluminium phosphates used according tothe invention will generally have a PZC of between 5.0 and 7.0, morepreferably between 5.5 and 6.0 e.g. about 5.7.

The aluminium phosphate is preferably used in the form of an aqueoussolution to which antigens are added (NB: it is standard to refer toaqueous aluminium phosphate as a “solution” although, on a strictphysicochemical view, the salt is insoluble and forms a suspension). Itis preferred to dilute the aluminium phosphate to the requiredconcentration and to ensure a homogenous solution before the addition ofthe antigenic components.

The concentration of Al³⁺ prior to addition of antigens is generallybetween 0 and 10 mg/ml. A preferred concentration is between 2 and 6mg/ml. A more preferred concentration is between 4 and 5 mg/ml e.g. 4.4mg/ml (corresponding to aluminium phosphate concentration of 20 mg/ml).The concentration of Al³⁺ in final vaccines of the invention isgenerally between 0.1 and 2.0 mg/ml. A preferred concentration isbetween 0.2 and 1.5 mg/ml. A more preferred concentration is between 0.3and 1.0 mg/ml. Most preferably, the concentration is about 0.6 mg/ml.

An aluminium phosphate solution used to prepare a vaccine of theinvention may contain a buffer (e.g. a phosphate or a histidine buffer),but this is not necessary. The aluminium phosphate solution ispreferably sterile and pyrogen-free. The aluminium phosphate solutionmay include free aqueous phosphate ions e.g. present at a concentrationbetween 1.0 and 20 mM, preferably between 5 and 15 mM, and morepreferably about 10 mM. The aluminium phosphate solution may alsocomprise sodium chloride. The concentration of sodium chloride ispreferably in the range of 0.1 to 100 mg/ml (e.g. 0.5-50 mg/ml, 1-20mg·ml, 2-10 mg/ml) and is more preferably about 3±1 mg/ml. The presenceof NaCl facilitates the correct measurement of pH prior to adsorption ofantigens.

Although the use of aluminium salts as sole adjuvants is normal, otheradjuvants which may be included in vaccines of the invention include,but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 65], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt [66].

Vaccine compositions of the invention are preferably substantially freeof aluminium hydroxides (e.g. aluminium oxyhydroxides). Theconcentration of aluminium hydroxides in the composition will usually beless than 100 μg/ml, preferably less than 50 μg/ml, more preferably lessthan 10 μg/ml, and most preferably less than 1 μg/ml. In particular, theHib-conjugate antigen is preferably not adsorbed onto an aluminiumhydroxide.

A calcium phosphate adjuvant may be used.

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 [Chapter 10 of ref. 65;see also ref. 67] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

C. Saponin Formulations [Chapter 22 of Ref. 65]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, sterns, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref. 68.Saponin formulations may also comprise a sterol, such as cholesterol[69].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs) [chapter 23 ofref. 65]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA and QHC. ISCOMs are further described in refs. 69-71. Optionally,the ISCOMS may be devoid of additional detergent [72].

A review of the development of saponin based adjuvants can be found inrefs. 73 & 74.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 75-80. Virosomes are discussed further in, for example, ref. 81

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 82. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [82]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [83, 84].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 85 & 86.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 87, 88 and 89 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 90-95.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [96]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 97-99. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 96 & 100-102.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 103 and as parenteraladjuvants in ref. 104. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 105-112. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 113, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [114], etc.) [115], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [116] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [117].

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref. 65)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 118-120.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [121]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [122] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [123]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 124 and 125.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 126 and 127.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [128]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL) [129]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) [130]; (5) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions [131]; (6) SAF,containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121,and thr-MDP, either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion. (7) Ribi™ adjuvant system(RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and oneor more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); and (8) one or more mineralsalts (such as an aluminum salt)+a non-toxic derivative of LPS (such as3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 65.

Further Antigens

Compositions of the invention contain D, T, P and Hib antigens. They mayalso include further antigens, such as:

-   -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 132 from        serogroup C or the oligosaccharides of ref. 133. The vaccine        preferably contains conjugates from 2, 3 or 4 of serogroups A,        C, W135 and Y.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. refs.        134 to 136].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 137, 138].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 138, 139].    -   an outer-membrane vesicle (OMV) or bleb preparation from N.        meningitidis serogroup B, such as those disclosed in refs. 140,        141, 142, 143, etc.    -   a protein antigen from N. meningitidis serogroup B, such as        those in refs. 144 to 150, with protein ‘287’ (see below) and        derivatives (e.g. ‘ΔG287’) being particularly preferred.    -   polio antigen(s) [e.g. 151, 152] such as IPV.

The composition may comprise one or more of these further antigens.Antigens will typically be present at a concentration of at least 1μg/ml each. In general, the concentration of any given antigen will besufficient to elicit an immune response against that antigen. It ispreferred that the protective efficacy of individual saccharide antigensis not removed by combining them, although actual immunogenicity (e.g.ELISA titres) may be reduced.

Where a saccharide antigen is used, it is preferably conjugated to acarrier protein in order to enhance immunogenicity.

As an alternative to using protein antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 153to 161]. Protein components of the compositions of the invention maythus be replaced by nucleic acid (preferably DNA e.g. in the form of aplasmid) that encodes the protein. Similarly, compositions of theinvention may comprise proteins which mimic saccharide antigens e.g.mimotopes [162] or anti-idiotype antibodies. These may replaceindividual saccharide components, or may supplement them. As an example,the vaccine may comprise a peptide mimic of the MenC [163] or the MenA[164] capsular polysaccharide in place of the saccharide itself.

Where a vaccine of the invention includes a hepatitis B surface antigen(‘HBsAg’), this antigen can be made in two ways. The first methodinvolves purifying the antigen in particulate form from the plasma ofchronic hepatitis B carriers, as large quantities of HBsAg aresynthesized in the liver and released into the blood stream during anHBV infection. The second way is preferred and involves expressing theprotein by recombinant DNA methods. It is preferred that the HBsAg isprepared by expression in the Saccharomyces cerevisiae yeast. The HBsAggene may be inserted into a plasmid, and its expression from the plasmidmay be controlled by a promoter such as the ‘GAPDH’ promoter (from theglyceraldehyde-3-phosphate dehydrogenase gene). The yeast may becultured in a synthetic medium. HBsAg can then be purified by a processinvolving steps such as precipitation, ion exchange chromatography, andultrafiltration. After purification, HBsAg may be subjected to dialysis(e.g. with cysteine). The HBsAg may be used in a particulate form.

Where a vaccine of the invention includes a polio antigen, it ispreferred to use three poliovirus antigens—poliovirus Type 1 (e.g.Mahoney strain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirusType 3 (e.g. Saukett strain). Polioviruses may be grown in cell culture.A preferred culture uses a VERO cell line, which is a continuous cellline derived from monkey kidney. VERO cells can conveniently be culturedmicrocarriers. Culture of the VERO cells before and during viralinfection may involve the use of bovine-derived material, such as calfserum, and this material should be obtained from sources which are freefrom bovine spongiform encephalitis (BSE). Culture may also involvematerials such as lactalbumin hydrolysate. After growth, virions may bepurified using techniques such as ultrafiltration, diafiltration, andchromatography. Prior to administration to patients, the viruses must beinactivated, and this can be achieved by treatment with formaldehyde.Viruses are preferably grown, purified and inactivated individually, andare then combined to give a bulk mixture for addition to the adsorbeddiphtheria and tetanus antigens.

Antigens in vaccines of the invention will be present in‘immunologically effective amounts’ i.e. the administration of thatamount to an individual, either in a single dose or as part of a series,is effective for treatment or prevention of disease. This amount variesdepending upon the health and physical condition of the individual to betreated, age, the taxonomic group of individual to be treated (e.g.human, primate, etc.), the capacity of the individual's immune system tosynthesise antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. Dosage treatment may be a single dose schedule or amultiple dose schedule (e.g. including booster doses).

Non-Immunological Components of Vaccines of the Invention

Vaccines of the invention will typically, in addition to the antigenicand adjuvant components mentioned above, comprise one or more‘pharmaceutically acceptable carriers’, which include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolised macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, sucrose [165], trehalose [166], lactose,and lipid aggregates (such as oil droplets or liposomes). Such carriersare well known to those of ordinary skill in the art. The vaccines mayalso contain diluents, such as water, saline, glycerol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in reference 167.

Compositions of the invention are in aqueous form i.e. solutions orsuspensions. Liquid formulation of this type allows the compositions tobe administered direct from their packaged form, without the need forreconstitution in an aqueous medium, and are thus ideal for injection.Compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses.

Liquid vaccines of the invention are also suitable for reconstitutingother vaccines from a lyophilised form. Where a vaccine is to be usedfor such extemporaneous reconstitution, the invention provides a kit,which may comprise two vials, or may comprise one ready-filled syringeand one vial, with the contents of the syringe being used to reactivatethe contents of the vial prior to injection.

Vaccines of the invention may be packaged in unit dose form or inmultiple dose form. For multiple dose forms, vials are preferred topre-filled syringes. Effective dosage volumes can be routinelyestablished, but a typical human dose of the composition for injectionhas a volume of 0.5 ml.

Vaccines of the invention generally have a pH of between 6.0 and 8.0,more preferably between 6.3 and 6.9 e.g. 6.6±0.2. Vaccines arepreferably buffered at this pH. Stable pH may be maintained by the useof a buffer. If a composition comprises an aluminium hydroxide salt, itis preferred to use a histidine buffer [168]. The composition may besterile and/or pyrogen-free. Aluminium phosphate and whole cellpertussis antigens are incompatible with filter sterilisation and so,when a composition of the invention includes one of these components, itis preferred to sterilise the composition of the invention byautoclaving and/or to use sterile components in its production.

Compositions of the invention may be isotonic with respect to humans.

Vaccines of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format. Many antimicrobials aremercury-based (e.g. thiomersal), although mercurial preservatives arepreferably avoided e.g. 2-phenoxyethanol may be used. Any preservativeis preferably present at low levels (e.g. 0.01% by volume). Preservativemay be added exogenously and/or may be a component of the bulk antigenswhich are mixed to form the composition (e.g. present as a preservativein pertussis antigens).

Vaccines of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%.

Vaccines of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. The composition may comprise sodiumchloride. The concentration of sodium chloride in the composition ispreferably in the range of 0.1 to 100 mg/ml (e.g. 1-50 mg/ml, 2-20mg/ml, 5-15 mg/ml) and is more preferably 10±2 mg/ml NaCl e.g. about 9mg/ml.

Vaccines of the invention will generally include a buffer. A phosphateor histidine buffer is typical.

Vaccines of the invention may include free phosphate ions in solution(e.g. by the use of a phosphate buffer) in order to favournon-adsorption of antigens. The concentration of free phosphate ions inthe composition of the invention is generally between 0.1 and 10.0 mM,preferably between 1 and 5 mM, and more preferably about 2.5 mM.

Packaging of Vaccines of the Invention

Vaccines of the invention can be packaged in various types of containere.g. in vials, in syringes, etc.

For prior art DTP-Hib vaccines that require reconstitution of alyophilised Hib component, aqueous DTP antigens are drawn from a firstsealed vial into a syringe and are then introduced into a second sealedvial that contains the lyophilised material. The reconstituted vaccineis then withdrawn into the same syringe for administration to a patient.In contrast, Hib conjugates in vaccines of the invention are stable inaqueous conditions and do not require lyophilisation. The invention isthus able to provide a vial having a piercable seal and containing aDTP-Hib vaccine, wherein the piercable seal has not been pierced.Similarly, the invention is able to provide a hermetically sealedcontainer containing a vaccine of the invention.

Prior art DTP-Hib vaccines that require reconstitution of a lyophilisedHib component must be packaged into two separate containers. Incontrast, the invention allows the provision of a process for insertinga vaccine of the invention into a container in a form that alreadyincludes DTP-Hib antigens. The insertion is preferably not through aseal in the container.

Similarly, as vaccine packaging is labelled during manufacture thenprior art vaccines DTP-Hib are labelled while they are in separate DTPand Hib formats, whereas the invention allows labelling of the vaccinein its final DTP-Hib format.

Furthermore, prior art reconstituted DTP-Hib vaccines are withdrawn fromtheir containers in DTP-Hib form but they are not inserted into thecontainers in that form. According to the invention, however, suchDTP-Hib vaccines can be both inserted and extracted in the admixedDTP-Hib form. The time between insertion and extraction can be at leastn weeks, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25 or more. Extraction will generally be via the needle (e.g. the needleof a sterile delivery device, such as a syringe), although insertioninto the container will usually be from a manufacturing line rather thanfrom a delivery device.

Preparing Vaccines of the Invention

Vaccines of the invention contain at least DTP-Hib antigens, and theirpreparation thus involves admixing these four antigens. Unlike thepreparation of reconstituted prior art DTP-Hib vaccines, the inventioncan provide a preparation process that does not include a step oflyophilisation of the Hib conjugate. Similarly, the invention is able toprovide a preparation process that does not include a step of packagingadmixed DTP antigens separately from Hib antigen.

During preparation, antigens which are added are generally not alreadyadsorbed onto an aluminium salt (i.e. they are not ‘pre-adsorbed’). Itis thus preferred that, for each of the antigens which is added, no morethan 5% by weight (preferably none) is already adsorbed to an aluminiumsalt (e.g. at most 4%, at most 3%, or at most 2%). In some situations,however, pre-adsorbed antigens may be added.

A typical process for preparing bulk vaccine of the invention will addthe Hib component to a mixture of the D, T and P components i.e. the DTPcomponents are mixed prior to addition of the Hib component. This orderof mixing allows the ionic strength and/or pH of the composition to beadjusted (e.g. pH<7) prior to addition of the Hib component in order toprevent adsorption to any aluminium adjuvant that may be present.

Vaccines of the invention are preferably prepared at between 15° C. and30° C. (e.g. between 19° C. and 27° C., or at 23±4° C.).

Administration of Vaccines of the Invention

The invention provides a method for raising an antibody response in amammal, comprising administering a vaccine of the invention to themammal. The vaccines can be administered prophylactically (i.e. toprevent infection) or therapeutically (i.e. to treat disease afterinfection).

The invention provides a method for raising an immune response in amammal, comprising the step of administering an effective amount of avaccine of the invention. The immune response is preferably protectiveand preferably involves antibodies. The method may raise a boosterresponse.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc.

The invention also provides compositions of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of at least diphtheria, tetanus,pertussis and H. influenzae type b (‘Hib’) antigens in the manufactureof a combined vaccine for the immunisation of a patient, wherein (a) theantigen for protecting against Hib is a conjugate of a Hib capsularsaccharide; (b) the concentration of the Hib conjugate in the vaccine is<15 μg/ml; and (c) the manufacture does not include lyophilisation ofthe Hib conjugate.

These uses and methods are preferably for the prevention and/ortreatment of C. diphtheriae, C. tetani, B. pertussis and H. influenzaeinfections and of disease caused by these infections e.g. the preventionof diphtheria, of tetanus, of whooping cough, of bacterial meningitis,etc.

One way of checking efficacy of therapeutic treatment involvesmonitoring bacterial infection after administration of the compositionof the invention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the antigens afteradministration of the composition. Immunogenicity of compositions of theinvention can be determined by administering them to test subjects (e.g.children 12-16 months age, or animal models [169]) and then determiningstandard immunological parameters. These immune responses will generallybe determined around 4 weeks after administration of the composition,and compared to values determined before administration of thecomposition. Rather than assess actual protective efficacy in patients,standard animal and in vitro models and correlates of protection forassessing the efficacy of Hib and DTP vaccines are well known.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

Bacterial infections affect various areas of the body and so thecompositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. The composition may be prepared forpulmonary administration e.g. as an inhaler, using a fine powder or aspray. The composition may be prepared as a suppository or pessary. Thecomposition may be prepared for nasal, aural or ocular administratione.g. as spray, drops, gel or powder [e.g. refs 170 & 171]. Successfulintranasal administration of Hib saccharides [172] and DTP vaccines[173, 174] has been reported.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows anti-PRP GMTs and 95% confidence intervals in the vaccinegroups A, B, C and D.

MODES FOR CARRYING OUT THE INVENTION

Four DTwP-Hib vaccine formulations were prepared, differing only intheir dose of Hib-CRM₉₇ conjugate. Vaccines were prepared as 0.5 mldoses with the following antigenic compositions:

A B C D [175] Hib-CRM197 conjugate (μg saccharide 1.25 2.5 5 10 perdose) Diphtheria toxoid (Lf per dose) 15 Tetanus toxoid (Lf per dose)3.2 Inactivated B. pertussis organisms (OU 15 per dose) Aluminiumphosphate adjuvant (mg Al³⁺ 0.3 per dose) NaCl (mg per dose) 4.5Merthiolate Trace Hib fraction relative to ref. 175 ⅛ ¼ ½ 1

The production process was essentially as follows: start with wfi; addthe aluminium phosphate adjuvant; add the D component; add the Tcomponent; add the wP component; add NaCl; check and adjust pH; and addthe Hib component. Contrary to the statement in reference 175, the Hibcomponent does not adsorb to the adjuvant.

Stability

Two stability studies were performed: one under normal storageconditions at 2-8° C. for 2 years, and another under acceleratedconditions at 37° C. for 14 days. Vaccines were tested after storage inan upright or an inverted position. Stability was assessed bydetermining pH and free saccharide.

Results of the first study were as follows:

RELATIVE HIB DOSE Months Parameter 1 ½ ¼ ⅛ 0 pH 6.7 6.5 6.5 6.6 % freesacc 2.9 <3.6 <7.2 <14.4 6 pH 6.8 6.7 6.7 6.7 % free sacc 2.6 <3.6 <7.2<14.5 12 pH 7.0 6.9 6.9 7.0 % free sacc 4.8 5.1 2.5 4.4 24 pH 7.1 7.07.0 7.1 % free sacc 6.1 5.8 5.6 5.0

Results of the accelerated stability study were as follows:

RELATIVE HIB DOSE Time Parameter 1 ½ ¼ ⅛ 0 pH 6.7 6.5 6.5 6.6 % freesacc 2.2 <3.6 <7.2 <14.4 14 days pH — — — — upright % free sacc 4.2 5.1<7.2 <14.5 14 days pH 6.8 6.7 6.7 6.8 inverted % free sacc 5.0 5.6 <7.2<14.5

The vaccines are thus stable over extended periods. Adsorption wastested over the same timescale, and the Hib conjugate remains unadsorbedduring storage.

Clinical Testing

A total of 261 infants were enrolled and randomised to receive one ofthe four vaccines in a double-blind trial to evaluate safety andimmunogenicity (cf. the studies in references 6, 8 and 9, which wereeither partially blinded or open). Three month-old infants in goodhealth, born at ≧37 weeks of gestation with a minimum birth weight of2500 g, and eligible for the local EPI, were enrolled and randomised ina 66:65:65:65 A:B:C:D ratio to receive three single intramuscular dosesof one of the vaccines at 3, 4 and 5 months of age. In accordance withthe local EPI, subjects received oral polio vaccine (OPV) in parallel at2, 4 and 6.

Mean age at enrolment was 94 (range 69-108) days and no difference wasobserved among the four vaccine groups in terms of sex, race, weight andheight distribution. Patients were followed for post-injection reactionsand adverse events. months of age. A total of 260 subjects were includedin the safety analyses (65 for each of the four vaccine groups), and 251in the immunogenicity analyses (61 in group A, 64 each in groups B andC, and 62 in group D). Nine subjects did not complete the study as theirparents/legal guardians withdrew their consent before study completion(5 subjects in group A, 1 subject in group B and 3 subjects in group D).

Blood samples were obtained at baseline and one month after the thirddose. Anti-PRP, anti-pertussis, anti-diphtheria and anti-tetanusantibodies were measured. IgG anti-PRP antibodies were measured by amodified ELISA adapted from the FDA ELISA method [176]. IgGanti-diphtheria toxin antibodies and IgG anti-tetanus toxin antibodywere quantified by ELISA. Serological markers for B. pertussis(anti-pertactin and anti-Agg2-3) were also measured by ELISA.

Safety and Reactogenicity

Subjects were monitored by study staff for 30 minutes after each vaccinedose. Parents were asked to record on a diary the daily rectaltemperature and local and systemic reactions for seven days followingeach injection. Additionally, study personnel actively contacted theparents/legal guardians by telephone on the second and seventh dayfollowing each vaccination to obtain particulars of any adverse events.Occurrence of adverse events, or serious adverse events and thosenecessitating physician visit and/or any medication were criticallyexamined and recorded throughout the study duration. Subjects whoreceived at least one vaccination were included in safety analyses.

Local and systemic reactions were mostly mild and transient. The mostfrequent were tenderness, erythema and induration for local reactions,and irritability, sleepiness and unusual crying for systemic reactions:

A: 1.25 μg B: 2.5 μg C: 5 μg D: 10 μg Local Tenderness - any 48 (74%) 53(82%) 52 (80%) 49 (75%) (Cried when injected limb was moved) 16 (25%) 19(29%) 16 (25%) 15 (23%) Erythema - any 31 (48%) 29 (45%) 37 (57%) 31(48%) (>50 mm) 6 (9%) 5 (8%)  7 (11%) 4 (6%) Induration - any 27 (42%)21 (32%) 25 (38%) 24 (37%) (>50 mm) 3 (5%) 4 (6%) 6 (9%) 3 (5%) SystemicRash 3 (5%) 4 (6%) 3 (5%) 3 (5%) Change in Eating Habits 20 (31%) 16(25%) 26 (40%) 27 (42%) Sleepiness 41 (63%) 49 (75%) 44 (68%) 39 (60%)Unusual Crying 29 (45%) 23 (35%) 32 (49%) 29 (45%) Persistent Crying 10(15%)  8 (12%) 10 (15%)  7 (11%) Irritability 47 (72%) 53 (82%) 50 (77%)49 (75%) Vomiting 5 (8%) 6 (9%)  9 (14%)  7 (11%) Diarrhoea 17 (26%) 24(37%) 22 (34%) 18 (28%) Other Rectal Temperature ≧38.5° C.-<40.5° C. 14(22%) 21 (32%) 10 (15%) 12 (18%) ≧40.5° C. 0 (0%) 0 (0%) 0 (0%) 0 (0%)Use of analgesic/antipyretic medication 33 (51%) 43 (66%) 28 (43%) 33(51%)

No significant differences were observed among the four vaccine groups,and all post-vaccination reactions resolved without sequelae. There wereno serious adverse events reported as related to study vaccines.

Immunogenicity

Anti-PRP seroconversion rates (% patients seroconverted) were asfollows:

1.25 μg 2.5 μg 5 μg 10 μg Titre ≧0.15 μg/ml ≧1.0 μg/ml ≧0.15 μg/ml ≧1.0μg/ml ≧0.15 μg/ml ≧1.0 μg/ml ≧0.15 μg/ml ≧1.0 μg/ml Pre 47% 12% 30%  6%32%  6%  34%  4% Post 98% 90% 97% 88% 98% 97% 100% 95%

Anti-pertussis seroconversion rates were assessed by looking at (a)increases of anti-pertactin and anti-Agg2-3 responses, and (b) GMTs.Results (% patients) were as follows:

1.25 μg 2.5 μg 5 μg 10 μg Anti-pertactin % patients with 2-fold increase91% 95% 88% 94% % patients with 4-fold increase 80% 83% 78% 89%Anti-pertactin GMT (μg/ml) 19  21  24  20  Anti-Agg2-3 % patients with2-fold increase 81% 81% 83% 80% % patients with 4-fold increase 70% 80%78% 71% Anti-Agg2-3 GMT (μg/ml) 87  77  66  76 

Anti-diphtheria and anti-tetanus GMTs were as follows:

1.25 μg 2.5 μg 5 μg 10 μg Anti-diphtheria GMT (μg/ml) 1.33 1.65 1.494.70 Anti-tetanus GMT (μg/ml) 4.89 5.10 5.24 5.16

The proportion of subjects with anti-PRP titres ≧0.15 μg/mL was thussimilar in the four vaccine groups, but the proportion of subjects withanti-PRP titres ≧1.0 μg/mL was higher in groups C and D (97% and 95%respectively) than in groups A and B (90% and 88% respectively).Anti-PRP GMTs show a clear dose-response effect, being similar betweengroups A (6.94 μg/ml) and B (7.82 μg/ml), and between groups C (17μg/mL) and D (18 μg/mL), but significantly lower in groups A and B whencompared with both C and D groups (FIG. 1).

One month after the third vaccination, all subjects in each of the fourvaccine groups seroconverted to diphtheria and tetanus (antibody level≧0.1 IU/mL). No appreciable differences were observed in GMTs among thefour vaccine groups.

No differences were observed among the four vaccine groups in theproportion of subjects with a 2- or 4-fold increase of anti-pertactinand anti-Agg2-3 antibodies compared with baseline. GMTs among the fourvaccination groups were also similar.

CONCLUSIONS

The DTwPHib vaccine formulations with fractional doses of 5, 2.5 or 1.25μg Hib-conjugate per dose were as immunogenic as the reference 10 μgformulation, as far as seroconversion rates were concerned. High andequivalent levels of anti-PRP GMTs were elicited by the formulationswith 10 μg or 5 μg of conjugate. Although formulations with 2.5 μg and1.25 μg of conjugate per dose elicited lower anti-PRP GMTs, they wereadequately immunogenic.

Thus the work confirms that the amount of Hib antigen can be reduced aslow as 1.25 μg per dose in an appropriate formulation containing DTPvaccines without affecting the protective efficacy of other vaccinecomponents.

All four formulations of the DTP-Hib vaccine were safe and immunogenicfor all vaccine components. In terms of anti-PRP seroprotection rates,the four formulations were equally immunogenic, both in the short term(titres ≧0.15 μg/mL) and in the long term (titres ≧1 μg/mL). Theformulation containing 5 μg conjugate was as immunogenic as thereference formulation containing 10 μg, and for both formulations theanti-PRP GMTs were particularly high (17 and 18 μg/mL). The formulationscontaining 1.25 and 2.5 μg conjugate were highly immunogenic whencompared with the immune responses observed in other studies usingfractional doses of Hib vaccines [6, 8, 9].

Further information can be found in reference 177.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES (THE CONTENTS OF WHICH ARE HEREBY INCORPORATED IN FULL)

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1. A combination vaccine comprising antigens for protecting a subjectagainst at least diphtheria (‘D’), tetanus (‘T’), pertussis (‘P’) andHaemophilus influenzae type b (‘Hib’), wherein: (a) the antigen forprotecting against Hib is a conjugate of a Hib capsular saccharide; (b)the concentration of the Hib conjugate in the vaccine is <15 μg/ml; and(c) the Hib conjugate has never been lyophilised.
 2. A vial having apiercable seal and containing a combination vaccine, which combinationvaccine comprises antigens for protecting a subject against at leastdiphtheria, tetanus, pertussis and H. influenzae type b (‘Hib’), whereinthe antigen for protecting against Hib is a conjugate of a Hib capsularsaccharide, and wherein: (a) the concentration of the Hib conjugate inthe vaccine is <15 μg/ml, and (b) the vial's piercable seal has not beenpierced.
 3. A hermetically-sealed container containing a combinationvaccine comprising antigens for protecting a subject against at leastdiphtheria, tetanus, pertussis and H. influenzae type b (‘Hib’), whereinthe antigen for protecting against Hib is a conjugate of a Hib capsularsaccharide, and wherein the concentration of the Hib conjugate in thevaccine is <15 μg/ml.
 4. A process for preparing a combination vaccinecomprising antigens for protecting a subject against at leastdiphtheria, tetanus, pertussis and H. influenzae type b (‘Hib’), whereinthe antigen for protecting against Hib is a conjugate of a Hib capsularsaccharide, and the concentration of Hib conjugate in the vaccine is <15μg/ml, characterised in that the process does not include one or both ofthe following steps (a) a step of lyophilisation of the Hib conjugateantigen; (b) a step of packaging the diphtheria, tetanus and pertussisantigens in admixed form separately from the Hib conjugate antigen.
 5. Aprocess for inserting a combination vaccine into a container, wherein:(a) the vaccine that comprises antigens for protecting a subject againstat least diphtheria, tetanus, pertussis and H. influenzae type b(‘Hib’); (b) the antigen for protecting against Hib is a conjugate of aHib capsular saccharide; and (c) the concentration of the Hib conjugatein the vaccine is <15 μg/ml.
 6. A process for attaching a label to acontainer, wherein: (a) the container contains a combination vaccinethat comprises antigens for protecting a subject against at leastdiphtheria, tetanus, pertussis and H. influenzae type b (‘Hib’); (b) theantigen for protecting against Hib is a conjugate of a Hib capsularsaccharide; and (c) the concentration of the Hib conjugate in thevaccine is <15 μg/ml.
 7. A process for inserting a combination vaccineinto a container and then extracting the vaccine from the container,wherein: (a) the vaccine comprises antigens for protecting a subjectagainst at least diphtheria, tetanus, pertussis and H. influenzae type b(‘Hib’); (b) the antigen for protecting against Hib is a conjugate of aHib capsular saccharide; and (c) the concentration of the Hib conjugatein the vaccine is <15 μg/ml.
 8. A combination vaccine comprisingantigens for protecting a subject against at least diphtheria (‘D’),tetanus (‘T’), pertussis (‘P’) and H. influenzae type b (‘Hib’),wherein: (a) the antigen for protecting against Hib is a conjugate of aHib capsular saccharide; (b) the concentration of the Hib conjugate inthe vaccine is <15 μg/ml; and (c) the vaccine (i) does not contain analuminium hydroxide adjuvant and/or (ii) does not contain an aluminiumpotassium sulphate adjuvant.
 9. The vaccine of claim 8, comprising analuminium phosphate adjuvant.
 10. The vaccine of claim 9, wherein theHib conjugate is not adsorbed to the aluminium phosphate adjuvant. 11.The vaccine, vial, container or process of any preceding claim, wherethe diphtheria antigen comprises a diphtheria toxoid, the tetanusantigen comprises a tetanus toxoid, and the pertussis antigen comprisesa cellular pertussis component.
 12. The vaccine, vial, container orprocess of any preceding claim, where the conjugate comprises a CRM₁₉₇carrier, a tetanus toxoid carrier or an outer membrane complex of N.meningitidis carrier.
 13. The vaccine, vial, container or process of anypreceding claim, where the conjugate comprises an oligosaccharidefragment of the Hib polyribosylribitol phosphate.
 14. The vaccine, vial,container or process of any preceding claim, wherein the combinationvaccine further comprises a surface antigen from hepatitis B virus. 15.The vaccine, vial, container or process of any preceding claim, whereinthe combination vaccine further comprises a polio antigen.
 16. A methodfor raising an antibody response in a mammal, comprising administeringthe vaccine of any preceding claim to the mammal.